Purpose:
To investigate the effects of tauroursodeoxycholic acid (TUDCA) and alpha-lipoic-acid (ALA) on the visual response properties of cat retinal ganglion cells (RGCs) in wholemount retinas.

Methods:
Young adult cats were divided into three groups: control, ALA, and TUDCA. In vitro single-unit extracellular recordings were performed on wholemount retinas to objectively evaluate the visual response properties of RGCs prior and post to antioxidant treatment. The visual responses properties of RGCs, including receptive field size, luminance threshold, and contrast sensitivity, were collected online and analyzed off-line with Axon Pclamp9.

Results:
Most of the RF sizes were larger than those plotted prior to the 60 minutes dark adaptation. The luminance threshold was elevated in the control group (no treatment) but reduced post ALA treatment and significantly reduced post TUDCA treatment. The contrast threshold was significantly elevated in the control group (no treatment) and clearly elevated post ALA treatment but effectively sustained post TUCDA treatment.

Conclusions:
Retinal neurocircuitry deteriorates in wholemount retinas, resulting in abnormal visual response properties in RGCs. Alpha-lipoic-acid and TUDCA exerted beneficial neuroprotective effects by activating the antioxidant pathway, partially restoring the functionality of retinal neurocircuitry and significantly improving the visual response properties of RGCs. However, TUDCA appears to be more effective than ALA in reducing irradiance thresholds and improving contrast sensitivity.

Retinal ganglion cells (RGCs) play a key role in integrating visual information within retinal neural circuits and relaying it to the visual centers of the brain. Analyzing the visual response properties of RGCs is an objective method that is used to assess the function of these neurons in the retinal neurocircuitry. Retinal ganglion cell death occurs in the early stages of glaucoma, in diabetic retinopathy, and in many other retinal diseases. Oxidative stress, induced by an increase in reactive oxygen species (ROS) and/or decreased antioxidant capacity, plays an important role in the pathogenesis of various retinal degenerative diseases, including diabetic retinopathy,1,2 glaucoma,3–5 age-related macular degeneration (AMD),6,7 retinitis pigmentosa,8,9 and aging.10–12 Superoxide dismutase (SOD) is an important component of antioxidant defense mechanisms in almost all living cells. There is a wealth of literature demonstrating that SOD plays a pivotal role in protecting injured RGCs in a variety of animal models of retinal disease.13–16 The generated ROS avidly interact with a large number of molecules, including other small inorganic molecules and proteins, lipids, carbohydrates, and nucleic acids. Recent results in our laboratory have shown that altered visual function in RGCs, characterized by reduced receptive field (RF) size, elevated luminance threshold, and attenuated contrast threshold, can be reversed by in vitro application of SOD.17 However, SOD is an enzyme, which makes it difficult to use as a therapeutic agent. Tauroursodeoxycholic acid (TUDCA), a potent inhibitor of apoptosis, acts by interfering with the upstream mitochondrial pathway that leads to cell death, inhibiting oxygen radical production, reducing endoplasmic reticulum (ER) stress, and stabilizing the unfolded protein response. Tauroursodeoxycholic acid exerts cytoprotective effects in a number of retinal degeneration models.18–20 Experimental evidence has shown that systemic injection of TUDCA effectively reduces ER stress, prevents apoptosis, and preserves cone functions in the Leber congenital amaurosis (LCA) animal model.21 However, little is known about the effects of TUDCA on the visual response properties of RGCs under excessive oxidative stress. To address this question, we designed an experiment that consisted of exposure to repeated dark adaptations and visual stimulations that ultimately resulted in extensive oxidative stress. Wholemount retinas were maintained in a recording chamber for more than 5 hours at room temperature (Fig. 1A). A series of physiological experiments were performed during this time to assess the visual function of the RGCs. The present study demonstrates that both TUDCA and alpha-lipoic-acid (ALA), a widely recognized metabolic antioxidant, improved the visual response properties of RGCs. However, TUDCA was better at improving the luminance and contrast sensitivities of RGCs.

Thirty young adult domestic cats (Felis catus) of either sex, ranging from 2 to 3 years in age and weighing between 2.5 and 3 kg, were used in these experiments. Animals were purchased from a local research animal provider and were housed under a 12-hour light/12-hour dark cycle, with food and water provided ad libitum. Animals were killed with overdose of sodium pentobarbital (150 mg/kg intravenously [IV]). All experiments were performed in accordance with the Peking University guidelines for animal research and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

In Vitro Preparation

The retinal preparation used in this study has been described previously.22,23 Briefly, the animal was dark adapted for 45 minutes prior to enucleation. Under dim red light, the lens and vitreous were carefully removed with a pair of fine-tip forceps. The eyecup was flat mounted, sclera side down, on the bottom of a recording chamber and then superfused with medium (Ames; Sigma-Aldrich Corp., St. Louis, MO, USA) at a fixed rate (5 mL/min) at room temperature (between 22°C and 24°C).

Visual Stimulation

Studies using computer-generated visual stimulation paradigms have been described previously.22,23 Briefly, visual stimuli were generated by programming a graphics card (Millennium 3000; Matrox, Dorval, Quebec, Canada), displayed on a 5-inch monochromic Cathode Ray Tube (CRT) monitor (P4 phosphor, 600 × 800 pixels, 60 Hz; SVGA; Kristel Corp., St. Charles, IL, USA), and imaged with a first-surface mirror and lens (Edmond Scientific, Barrington, NJ, USA) on the film plane of the microscope's camera port. This method ensured that the stimulus was sharply focused when the electrode tip was in focus with the eyepiece. The luminance level of the CRT was measured by placing a silicon detector head perpendicular to the CRT surface (model 260-CRT; UDT Instruments, San Diego, CA, USA). The detector was linked to a digital radiometer (S370; UDT Instruments). The output of the radiometer was sent to a personal computer. The maximum luminance was 183 cd/m2. Background luminance levels on the CRT in the absence of stimulation were close to 0.4 cd/m2. During this experiment, a 40× water-immersion objective lens was used (Carl Zeiss Meditec, Inc., Thornwood, NY, USA). With this objective, the maximum luminance was further reduced to 0.69 cd/m2 on the retinal surface. The irradiance of visual stimuli at the retinal surface was determined by placing the sensor directly under the objective lens. The total irradiance (integrated over wavelength) arriving at the retina was calculated based on the assumption that, at 500 nm, 1 cd/m2 = 1 lumen/m2 = 683−1 × 25.15 × 1017/m2/s = 3.68 × 1015/m2/s = 3.68 × 103 photons/μm2/s. Thus, the unattenuated total irradiance was equal to 0.69 × 3.68 × 103 = 2.54 × 103 photons/μm2/s. This irradiance was further reduced by using different combinations of neutral-density filters (Oriel Corp., Stratford, CT, USA).

Recording Arrangement and Antioxidant Application

The experimental procedures are shown in Figure 1A. Briefly, a pre-recording acclimation period of 60 minutes was followed by a mapping of the RF and then a determining of the area threshold. After a 40-minute dark adaptation, luminance sensitivity was determined, followed by a contrast threshold measurement. Next, the antioxidant TUDCA (580549, Merck Millipore, Calbiochem, USA, concentration: 0.5 μM; volume: 25 mL; delivery rate: 5 mL/min; duration: 5 minutes) or ALA (T1395, Sigma-Aldrich Corp., concentration: 0.5 μM; volume: 25 mL; delivery rate: 5 mL/min; duration: 5 minutes) was delivered to the recording chamber, and the retina was dark adapted again for 60 minutes. The above procedures were then repeated. For the control group, no antioxidants were applied to the perfusion system. The entire experiment lasted for at least 5 hours.

Physiological Recording and Data Analysis

Visual responses were recorded extracellularly using a glass microelectrode (TW150F-4; World Precision Instruments, Sarasota, FL, USA), amplified with an intracellular amplifier (IR283; Neurodata, Inc., Delaware Water, PA, USA), and digitized with a data acquisition system (Digidata 1440; Axon Instruments, Inc., Forest City, CA, USA). The recording electrode was filled with 3 M NaCI. After visualization with the nuclear stain acridine orange (0.002%; Sigma-Aldrich Corp.), cells that had large soma were selected for recording. The receptive field was mapped with a 0.2° light spot. The spot was turned on and off while it was moved to various regions within the RF. Once the polarity of the RF was established, the spot was moved to a peripheral region so that the edge of the RF field could be determined. An area-threshold visual stimulation program was performed to determine the stimulus size that evoked the maximum discharge. The spot size that evoked the maximum response in the area threshold test was selected for luminance sensitivity analysis. Retinas were dark adapted for 40 minutes before luminance sensitivity tests. The intensity of the testing spot was attenuated by a series of neutral-density filters (Oriel Corp.). Incremental threshold luminance sensitivity tests were conducted according to previously published methods.17,23 In general, a criterion response for threshold irradiance was obtained by gradually increasing the intensity levels of the testing stimuli; a firing rate of two spikes per second above the baseline rate was set as a threshold response. If none of the test stimuli produced exactly two spikes per second, the threshold was determined by linear interpolation. This difference also matched our subjective auditory criteria. For contrast sensitivity testing, we applied conventional Fourier analysis techniques to plot poststimulus time histograms and to determine the amplitude of response components at the frequency of stimulation (fundamental) and at the second harmonic. This amplitude was used to estimate a cell's responsivity. Histograms were based on unit responses to a minimum of eight stimulus cycles. Axon digital scope 10 was used to acquire the data. The acquired data were further analyzed off-line (pCLAMP9 software; Axon Corp., San Mateo, CA, USA). Additional data analysis, including fast Fourier analysis and ANOVA, was performed with Microsoft Excel (Microsoft Corp., Redmond, WA, USA).

Results

Experimental Procedures

The experiment consisted of four steps (Fig. 1A). First, a pre-recording step of 60 minutes included eyecup preparation, transfer of the wholemount retina to a recording chamber, and time to allow the retina to stabilize in the perfusion system. This was followed by a pretreatment physiological evaluation time of 90 minutes, which includes mapping of the RF and determining RF size by area threshold test (15 minutes). After 40 minutes of dark adaptation (arrow head), irradiance and contrast threshold tests were performed (35 minutes). At the end of the first test round, antioxidants were delivered to the perfusion system (open arrow, 5 minutes). The retina was dark adapted for 60 minutes (large arrow head) prior to the next round of 90 minutes posttreatment visual function evaluation. The entire round of physiological testing lasted at least 5 hours. Figures 1B and 1D reveal a normalized irradiance threshold response during the 5-hour incubation period. Figures 1C and 1E show a normalized threshold response during the same period. As shown in Figure 2, after the pre-recording period, the RF center was mapped with a 0.2° testing spot. An ON-center cell (217-C1) was mapped first (Fig. 2A), and the estimated RF size was assessed by the area threshold test (Fig. 2B). The luminance threshold was examined after 40 minutes of dark adaptation (Figs. 2C1, 2D). This was followed by a spatial frequency tuning test to select the best spatial frequency for the next round of the contrast threshold experiment. Figure 2E shows the discharge patterns of the cell to sinusoidal grating (0.24 cyc/deg, 100% contrast, Fig. 2E1). This was followed by a contrast threshold test (Fig. 2F). Because this cell served as a control, no antioxidant was applied. The cell was allowed to dark adapt for 60 minutes, followed by the next round of visual function tests, which reexamined the RF size (Fig. 2A), the luminance threshold post 40 minutes of dark adaptation (Figs. 2C2, 2D), and the contrast threshold (Figs. 2E2, 2F) of the cell.

The receptive field was mapped, and an area threshold test was then performed to determine the RF center. As summarized in Table 1, six cells were recorded (four ON [light on visual stimulation evoked responses], two OFF [light off visual stimulation evoked responses] cells) in the control group, seven cells were recorded in the ALA group (all ON cells), and 12 cells were recorded in the TUDCA group (10 ON and two OFF cells). Figure 3A shows the RF center size change of a RGC (217-C1) in control group during 5 hours of incubation in the recording chamber. In the control group, the average RF size of the recorded cell increased from 1.86° to 2.26° (paired t test, P = 0.118, SEM = 0.214, n = 6). Figure 3B reveals the RF center size change of a RGC (122-C2) in the ALA group. On average, the RF size of the recorded cells was 2.11° prior to ALA application and 2.31° after; this size change was not significant (paired t test, P = 0.082, SEM = 0.095, n = 7). Figure 3C shows RF size change of a RGC (115-C1) prior and post to TUDCA treatment. The average RF increased from 1.97° prior to the treatment to 2.26° after the treatment. TUDCA treatment significantly increased the average RF size of recorded cells (Fig. 3D; paired t test, P < 0.001, SEM = 0.053, n = 12). However, there were no significant changes in the difference of RF size changes (Δ RF) between control and treatment groups (Fig. 3E; control to ALA [ANOVA, P = 0.374]; control to TUDCA [ANOVA, P = 0.517]; ALA to TUDCA [AVOVA, P = 0.351]).

The optimal spot size, which evoked the maximum discharge, was selected to determine the luminance threshold (Fig. 2B). This stimulus was used in conjunction with at least six neutral density filters that were placed in the optic path to measure threshold irradiance. At 3.4 log photons/μm2/s, cell 217-C1 showed vibrant discharges (Fig. 4A1). However, reducing the irradiance approximately 10-fold (2.4 log photons/μm2/s) yielded a substantial reduction in firing rate (Fig. 4A3). The impulse rates of cell 122-C2 (Fig. 4B1) and cell 115-C1 (Fig. 4C1) were also strongly modulated by testing spots, and reduced irradiance levels substantially decreased discharge rates (Figs. 4B3, 4C3). Response profiles are shown (Fig. 4A5). This cell in the control retina exhibited a substantially reduced response magnitude at all tested irradiance levels. It could therefore be driven by a wide range of visual stimulus intensity, whereas extended maintenance in the recording chamber (more than 3 hours) resulted in the production of responses only to high-intensity visual stimuli. As summarized in Table 2, this response pattern was confirmed in most recorded cells in later experiments. In contrast, after antioxidants were delivered to the perfusion system, both cells exhibited substantially elevated firing rates at both irradiance levels. The irradiance response profiles of two additional cells are shown in Figures 4B5 (122-C2) and 4C5 (115-C1), respectively. It is evident that cells exhibited substantially elevated irradiance thresholds without antioxidant treatment (Fig. 4A5), that ALA treatment reduced the irradiance threshold to the basal level (Fig. 4B5), and that TUDCA substantially reduced the irradiance threshold of the cell to a point surpassing its baseline threshold (Fig. 4C5). We observed a statistically significant elevation in the threshold irradiance in cells recorded after prolonged maintenance in the recording chamber, compared to normal control cells (Fig. 4D; paired t-test; P = 0.029, SEM = 0.082, n = 6). However, as shown in Figure 1B and Table 2, the ALA treatment reduced the irradiance threshold (Fig. 4D; paired t-test; P < 0.459, SEM = 0.075, n = 7), and the TUDCA treatment greatly elevated the irradiance sensitivity of the recorded cells (Fig. 4D; paired t-test; P < 0.001, SEM = 0.0258, n = 12). This trend of the irradiance threshold suppression by the TUDCA is shown in Figure 1D. The difference of average threshold irradiance (Δ threshold irradiance) was significantly reduced after both ALA (Fig. 4E; ANOVA, P = 0.019) and TUDCA (Fig. 4E; ANOVA, P < 0.001) treatments. There was, however, no statistically significant difference between ALA and TUDCA treated cells (Fig. 4E; ANOVA, P = 0.177).

Irradiance threshold prior to and post antioxidant treatment. (A) Depicts the discharge patterns of a recorded RGC (217-C1 ON, control) in response to two different levels of irradiance stimulation prior to (A1 [3.4 log photons/μm2/s] and A3 [2.43 log photons/μm2/s]) and post antioxidant treatment (A2 [3.4 log photons/μm2/s] and A4 [2.43 log photons/μm2/s]). This cell served as a control; it received no treatment. (A5) Reveals the irradiance response profile of the cell. The filled triangles and solid line depict the irradiance response profile after the first 40 minutes of the dark adaption. The open circles and dashed line show the response profile after the second 40 minutes of the dark adaption. The vertical coordinate is response magnitude (spikes/s). The abscissa shows irradiance levels (log photons/μm2/s). (B) Shows the discharge patterns of a recorded RGC in response to two different levels of irradiance stimulation (122-C2 ON, ALA) prior to (B1 [3.4 log photons/μm2/s] and B3 [0.87 log photons/μm2/s]) and post ALA treatment (B2 [3.4 log photons/μm2/s] and B4 [0.87 log photons/μm2/s]). (B5) Reveals the irradiance response profile of the cell. (C) Reveals the discharge patterns of a recorded RGC (115-C1 ON, TUDCA) in response to two different levels of irradiance stimulation prior to (C1 [3.34 log photons/μm2/s] and C3 [1.85 log photons/μm2/s]) and post TUDCA treatment (C2 [3.34 log photons/μm2/s] and C4 [1.85 log photons/μm2/s]). (C5) Reveals the irradiance response profile of the cell prior and post to TUDCA treatment. (D) A histogram comparing averaged irradiance thresholds under control, ALA, and TUDCA conditions. (E) A histogram comparing the range of averaged irradiance threshold differences between control, ALA, and TUDCA treated cells. Other conventions are as for Figure 2.

Figure 4

Irradiance threshold prior to and post antioxidant treatment. (A) Depicts the discharge patterns of a recorded RGC (217-C1 ON, control) in response to two different levels of irradiance stimulation prior to (A1 [3.4 log photons/μm2/s] and A3 [2.43 log photons/μm2/s]) and post antioxidant treatment (A2 [3.4 log photons/μm2/s] and A4 [2.43 log photons/μm2/s]). This cell served as a control; it received no treatment. (A5) Reveals the irradiance response profile of the cell. The filled triangles and solid line depict the irradiance response profile after the first 40 minutes of the dark adaption. The open circles and dashed line show the response profile after the second 40 minutes of the dark adaption. The vertical coordinate is response magnitude (spikes/s). The abscissa shows irradiance levels (log photons/μm2/s). (B) Shows the discharge patterns of a recorded RGC in response to two different levels of irradiance stimulation (122-C2 ON, ALA) prior to (B1 [3.4 log photons/μm2/s] and B3 [0.87 log photons/μm2/s]) and post ALA treatment (B2 [3.4 log photons/μm2/s] and B4 [0.87 log photons/μm2/s]). (B5) Reveals the irradiance response profile of the cell. (C) Reveals the discharge patterns of a recorded RGC (115-C1 ON, TUDCA) in response to two different levels of irradiance stimulation prior to (C1 [3.34 log photons/μm2/s] and C3 [1.85 log photons/μm2/s]) and post TUDCA treatment (C2 [3.34 log photons/μm2/s] and C4 [1.85 log photons/μm2/s]). (C5) Reveals the irradiance response profile of the cell prior and post to TUDCA treatment. (D) A histogram comparing averaged irradiance thresholds under control, ALA, and TUDCA conditions. (E) A histogram comparing the range of averaged irradiance threshold differences between control, ALA, and TUDCA treated cells. Other conventions are as for Figure 2.

Contrast threshold responses prior to and post antioxidant treatment. (A) Depicts the discharge patterns of a recorded RGC (217-C1 ON, control) in response to two different levels of contrast stimulation prior to (A1 [100%] and A3 [40%]) and post antioxidant treatment (A2 [100%] and A4 [40%]). This cell served as a control; it received no treatment. (A5) Reveals the contrast response profiles of the cell. Filled triangles and solid line depict the contrast response profile after the first 40 minutes of dark adaption. Open circles and dashed line show the response profile after the second 40 minutes of dark adaption. The vertical coordinate is response magnitude (spikes/s). The abscissa shows contrast levels (%). (B) Shows the discharge patterns of a recorded RGC in response to two different levels of contrast stimulation (122-C2 ON, ALA) prior to (B1 [100%] and B3 [10%]) and post ALA treatment (B2 [100%] and B4 [10%]). (B5) Reveals the contrast response profiles of the cell. (5C) Reveals the discharge patterns of a recorded RGC in response to two different levels of contrast stimulation (115-C1 ON, TUDCA) prior to (C1 [100%] and C3 [20%]) and post TUDCA treatment (C2 [100%] and C4 [20%]). (C5) Reveals the contrast response profile of the cell prior to and post the TUDCA treatment. (D) A histogram comparing averaged contrast thresholds under control, ALA, and TUDCA conditions. (E) A histogram comparing the range of averaged contrast threshold differences between control, ALA treated, and TUDCA treated cells. Other conventions are as for Figure 2.

Figure 5

Contrast threshold responses prior to and post antioxidant treatment. (A) Depicts the discharge patterns of a recorded RGC (217-C1 ON, control) in response to two different levels of contrast stimulation prior to (A1 [100%] and A3 [40%]) and post antioxidant treatment (A2 [100%] and A4 [40%]). This cell served as a control; it received no treatment. (A5) Reveals the contrast response profiles of the cell. Filled triangles and solid line depict the contrast response profile after the first 40 minutes of dark adaption. Open circles and dashed line show the response profile after the second 40 minutes of dark adaption. The vertical coordinate is response magnitude (spikes/s). The abscissa shows contrast levels (%). (B) Shows the discharge patterns of a recorded RGC in response to two different levels of contrast stimulation (122-C2 ON, ALA) prior to (B1 [100%] and B3 [10%]) and post ALA treatment (B2 [100%] and B4 [10%]). (B5) Reveals the contrast response profiles of the cell. (5C) Reveals the discharge patterns of a recorded RGC in response to two different levels of contrast stimulation (115-C1 ON, TUDCA) prior to (C1 [100%] and C3 [20%]) and post TUDCA treatment (C2 [100%] and C4 [20%]). (C5) Reveals the contrast response profile of the cell prior to and post the TUDCA treatment. (D) A histogram comparing averaged contrast thresholds under control, ALA, and TUDCA conditions. (E) A histogram comparing the range of averaged contrast threshold differences between control, ALA treated, and TUDCA treated cells. Other conventions are as for Figure 2.

The present results suggest that retinal neurocircuitry deteriorates following extended continuous in vitro recording in wholemount retinas. This leads, in turn, to abnormal visual response characteristics in RGCs. Both ALA and TUDCA exerted beneficial neuroprotective effects via the activation of the antioxidant pathway, partially restored functionality in retinal neurocircuitry, and improved the visual response properties of RGCs. However, under the present experimental condition, TUDCA, a potent antioxidant agent, offered superior antioxidant effects than ALA and quickly scavenges ROS from retinal neural circuits and significantly protected visual functions of RGCs.

ALA and TUDCA Promote RGC Visual Functions

Oxidative stress plays a crucial role in various retinal diseases including glaucoma, diabetic retinopathy, and AMD. Alpha-lipoic-acid and TUCDA have been shown to have neuroprotective effects through their antioxidant activity. However, most experiments were designed to either systematically evaluate the long-term effects of these antioxidants on retinal neural structural properties and gene expression or oxidative stress-induced cellular changes in cultured neurons. Few studies have evaluated the impact of these antioxidants on retinal visual functions using an electroretinogram (ERG).24–27 The visual response properties of individual RGCs in response to these antioxidants treatments have yet to be studied. The present study investigated the effects of ALA and TUDCA on the visual functions of single RGCs. It was observed that as shown in Figure 1B, incubation in the Ames medium for 5 hours elevated the irradiance threshold and that the ALA treatment did not prevent the threshold elevation. Tauroursodeoxycholic acid, however, effectively suppressed the irradiance threshold elevation (Fig. 1D). Furthermore, as shown in Figure 1C, the ALA treatment could not effectively offset the contrast threshold elevation, but TUDCA successfully minimized the contrast threshold elevation (Fig. 1E). Although the long-term effects of both antioxidants on RGCs have not been evaluated, the present results suggest that TUDCA is more effective in protecting RGC visual functions than is ALA, at least within 3 hours after the treatment. Therefore, the following discussion will focus on the impact of TUDCA on retinal neurocircuitry and the visual function of RGCs.

TUDCA Impact on Retinal Neurocircuitry

Tauroursodeoxycholic acid has been widely used as a potent inhibitor of apoptosis. It prevents oxygen radical production, reduces ER stress, and stabilizes the unfolded protein response. Tauroursodeoxycholic acid exerts cytoprotective effects in a number of retinal degeneration models.18–20 However, most experimental evidence has been derived from studies using systemic application of the antioxidant. For example, in animal models, a daily intraperitoneal injection of TUDCA (500 mg/kg) had anti-inflammatory effects,28 preserved photoreceptors after retinal detachment,29 and significantly reduced loss of rod and cone function after exposure to bright light.30 Similarly, subcutaneous injection of TUDCA (500 mg/kg) was effective in reducing ER stress, preventing apoptosis, and preserving cone function in the Leber congenital amaurosis animal model.21 Tauroursodeoxycholic acid also rescued ERG b-waves and the outer nuclear layer in an animal model of retinal degeneration.31 The dosage used in most in vitro experiments is much lower; for example, 100 μM of TUDCA added to culture medium effectively protected retinal neural cell cultures from cell death induced by elevated glucose concentration, decreased the mito-nuclear translocation of apoptosis-inducing factor,32 and suppressed expression of p-c-Jun and p-JNK in diabetic rat retinas and retinas exposed to high glucose.33 However, little is known about the dosage and direct effects of TUDCA on the visual response properties of RGCs under excessive oxidative stress. We report here, for the first time, on the impact of TUDCA (0.5 μM concentration [volume: 25 mL; deliver rate: 5 mL/min; duration: 5 minutes]) on the visual response properties of RGCs recorded from wholemount retinas. Retinas were perfused with oxygenated medium for at least 5 hours. In comparison to the control and ALA treatments, TUDCA effectively protected retinal neurocircuitry and maintained visual function in RGCs. Specifically, TUDCA treatment significantly enlarged RF size (Fig. 3D), reduced irradiance threshold (Fig. 4D), and maintained contrast threshold at the same level as before the treatment (Fig. 5D).

TUDCA Affects Visual Function in RGCs

We encountered more ON-center cells than OFF-center cells in both the controls and antioxidant treated retinas (Table 1; control: six cells, four ON and two OFF; ALA: six cells, all ON; TUDCA: 10 ON and two OFF). We could not exclude the possibility that our recording method may have had a sampling bias. However, using the same recording setup and approach, we demonstrated in congenic and dystrophic RCS rats that sampling bias had only a limited impact.22,23 If sampling bias is discounted, the present data suggest that OFF cells absorb more impact from exposure to antioxidants than ON cells, likely due to the long incubation time in the recording chamber. It is possible that the retina may become hypoxic in the dark-adapted state due to the large energy requirement of rods.34 This energy consumption could further diminish the already low oxygen tension of the inner plexiform layer (IPL), which is one of the highest O2 consumption regions in the retina.35,36 Evidence also shows that processes occurring in the deeper regions of IPL that are involved in the OFF pathway have higher oxygen demands.37 Thus, OFF cells may be more vulnerable to hypoxic attack than ON cells. Because we assessed the same cells before and after the antioxidant treatment, we could not determine whether we would encounter more ON or OFF cells after the TUDCA treatment. We did observe that RF size increased after treatment, but because only a few OFF cells were recorded, it remains to be determined whether TUDCA exerts the same protections in ON and OFF cells. Nevertheless, in db/db mice, we did not observe a significant change in the average RF size of ON cells after SOD application, whereas the RF size of OFF cells was significantly increased in these mice.17 The contrast response of ON and OFF cells was also affected differently by SOD treatment in db/db mice: the contrast threshold of ON cells was not affected by the SOD application, while the contrast threshold of OFF cells was dramatically suppressed. Our results reveal that contrast threshold of RGCs, recorded primarily in ON-center cells, was substantially improved after the TUDCA treatment (Fig. 5D). However, due to small sample size, particularly of control group, additional experiment needs to be performed to confirm current observations.

Together, these findings may have relevance in the treatment of retinal degenerative diseases, and TUDCA may be used as an effective therapeutic agent for preventing vision loss in metabolically stressed retinal diseases.

Acknowledgments

Supported by the National Basic Research Program of China (2011CB510206 [MP] and 2012CB825503 [JG]), Beijing Municipal Science & Technology Commission [MP], and the National Science Foundation of China (30831160516 [MP]).

Ames
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Irradiance threshold prior to and post antioxidant treatment. (A) Depicts the discharge patterns of a recorded RGC (217-C1 ON, control) in response to two different levels of irradiance stimulation prior to (A1 [3.4 log photons/μm2/s] and A3 [2.43 log photons/μm2/s]) and post antioxidant treatment (A2 [3.4 log photons/μm2/s] and A4 [2.43 log photons/μm2/s]). This cell served as a control; it received no treatment. (A5) Reveals the irradiance response profile of the cell. The filled triangles and solid line depict the irradiance response profile after the first 40 minutes of the dark adaption. The open circles and dashed line show the response profile after the second 40 minutes of the dark adaption. The vertical coordinate is response magnitude (spikes/s). The abscissa shows irradiance levels (log photons/μm2/s). (B) Shows the discharge patterns of a recorded RGC in response to two different levels of irradiance stimulation (122-C2 ON, ALA) prior to (B1 [3.4 log photons/μm2/s] and B3 [0.87 log photons/μm2/s]) and post ALA treatment (B2 [3.4 log photons/μm2/s] and B4 [0.87 log photons/μm2/s]). (B5) Reveals the irradiance response profile of the cell. (C) Reveals the discharge patterns of a recorded RGC (115-C1 ON, TUDCA) in response to two different levels of irradiance stimulation prior to (C1 [3.34 log photons/μm2/s] and C3 [1.85 log photons/μm2/s]) and post TUDCA treatment (C2 [3.34 log photons/μm2/s] and C4 [1.85 log photons/μm2/s]). (C5) Reveals the irradiance response profile of the cell prior and post to TUDCA treatment. (D) A histogram comparing averaged irradiance thresholds under control, ALA, and TUDCA conditions. (E) A histogram comparing the range of averaged irradiance threshold differences between control, ALA, and TUDCA treated cells. Other conventions are as for Figure 2.

Figure 4

Irradiance threshold prior to and post antioxidant treatment. (A) Depicts the discharge patterns of a recorded RGC (217-C1 ON, control) in response to two different levels of irradiance stimulation prior to (A1 [3.4 log photons/μm2/s] and A3 [2.43 log photons/μm2/s]) and post antioxidant treatment (A2 [3.4 log photons/μm2/s] and A4 [2.43 log photons/μm2/s]). This cell served as a control; it received no treatment. (A5) Reveals the irradiance response profile of the cell. The filled triangles and solid line depict the irradiance response profile after the first 40 minutes of the dark adaption. The open circles and dashed line show the response profile after the second 40 minutes of the dark adaption. The vertical coordinate is response magnitude (spikes/s). The abscissa shows irradiance levels (log photons/μm2/s). (B) Shows the discharge patterns of a recorded RGC in response to two different levels of irradiance stimulation (122-C2 ON, ALA) prior to (B1 [3.4 log photons/μm2/s] and B3 [0.87 log photons/μm2/s]) and post ALA treatment (B2 [3.4 log photons/μm2/s] and B4 [0.87 log photons/μm2/s]). (B5) Reveals the irradiance response profile of the cell. (C) Reveals the discharge patterns of a recorded RGC (115-C1 ON, TUDCA) in response to two different levels of irradiance stimulation prior to (C1 [3.34 log photons/μm2/s] and C3 [1.85 log photons/μm2/s]) and post TUDCA treatment (C2 [3.34 log photons/μm2/s] and C4 [1.85 log photons/μm2/s]). (C5) Reveals the irradiance response profile of the cell prior and post to TUDCA treatment. (D) A histogram comparing averaged irradiance thresholds under control, ALA, and TUDCA conditions. (E) A histogram comparing the range of averaged irradiance threshold differences between control, ALA, and TUDCA treated cells. Other conventions are as for Figure 2.

Contrast threshold responses prior to and post antioxidant treatment. (A) Depicts the discharge patterns of a recorded RGC (217-C1 ON, control) in response to two different levels of contrast stimulation prior to (A1 [100%] and A3 [40%]) and post antioxidant treatment (A2 [100%] and A4 [40%]). This cell served as a control; it received no treatment. (A5) Reveals the contrast response profiles of the cell. Filled triangles and solid line depict the contrast response profile after the first 40 minutes of dark adaption. Open circles and dashed line show the response profile after the second 40 minutes of dark adaption. The vertical coordinate is response magnitude (spikes/s). The abscissa shows contrast levels (%). (B) Shows the discharge patterns of a recorded RGC in response to two different levels of contrast stimulation (122-C2 ON, ALA) prior to (B1 [100%] and B3 [10%]) and post ALA treatment (B2 [100%] and B4 [10%]). (B5) Reveals the contrast response profiles of the cell. (5C) Reveals the discharge patterns of a recorded RGC in response to two different levels of contrast stimulation (115-C1 ON, TUDCA) prior to (C1 [100%] and C3 [20%]) and post TUDCA treatment (C2 [100%] and C4 [20%]). (C5) Reveals the contrast response profile of the cell prior to and post the TUDCA treatment. (D) A histogram comparing averaged contrast thresholds under control, ALA, and TUDCA conditions. (E) A histogram comparing the range of averaged contrast threshold differences between control, ALA treated, and TUDCA treated cells. Other conventions are as for Figure 2.

Figure 5

Contrast threshold responses prior to and post antioxidant treatment. (A) Depicts the discharge patterns of a recorded RGC (217-C1 ON, control) in response to two different levels of contrast stimulation prior to (A1 [100%] and A3 [40%]) and post antioxidant treatment (A2 [100%] and A4 [40%]). This cell served as a control; it received no treatment. (A5) Reveals the contrast response profiles of the cell. Filled triangles and solid line depict the contrast response profile after the first 40 minutes of dark adaption. Open circles and dashed line show the response profile after the second 40 minutes of dark adaption. The vertical coordinate is response magnitude (spikes/s). The abscissa shows contrast levels (%). (B) Shows the discharge patterns of a recorded RGC in response to two different levels of contrast stimulation (122-C2 ON, ALA) prior to (B1 [100%] and B3 [10%]) and post ALA treatment (B2 [100%] and B4 [10%]). (B5) Reveals the contrast response profiles of the cell. (5C) Reveals the discharge patterns of a recorded RGC in response to two different levels of contrast stimulation (115-C1 ON, TUDCA) prior to (C1 [100%] and C3 [20%]) and post TUDCA treatment (C2 [100%] and C4 [20%]). (C5) Reveals the contrast response profile of the cell prior to and post the TUDCA treatment. (D) A histogram comparing averaged contrast thresholds under control, ALA, and TUDCA conditions. (E) A histogram comparing the range of averaged contrast threshold differences between control, ALA treated, and TUDCA treated cells. Other conventions are as for Figure 2.